Infection and invasion by influenza viruses requires the intermediacy of sialic acid residues on the surface of the host cell. The terms sialic acid and neuraminic acid are used interchangeably. Similarly, sialidase and neuraminidase (NA) are used interchangeably. Initial attachment of the virus to the host cell occurs via the binding of the virus to these sialic acids (charged, 9-carbon sugars) through the hemagglutinin protein of the virus. Once inside the cell the virus replicates by taking advantage of the host cellular machinery. However, in order to remain optimally infective, the virus has evolved an NA that cuts off the sialic acid from the host cell surface to assist the virus in escaping the host cell to infect other cells. Failure to cut off the sialic acid from the host cell surface, results in retention of virus through attachment to the host cell.
The GH33 family of neuraminidases contains all the sialidases except the viral enzymes (GH34 family). The GH33 and GH34 families are distinct structurally and by sequence (See Cantarel B L. et al. (2009); and Henrissat B. and Davies G J (1997) for background on Family classifications). Previous work has demonstrated that 2,3-difluorosialic acids (DFSAs) are effective inhibitors of GH33 NAs and that GH33 NAs proceed through a covalent intermediate (see for example, Watts, A. et al. (2003); Amaya, M. F. et al. (2004); Watts, A. G. and Withers, S. G. (2004); Watts, A. G. et al. (2006); Newstead, S. et al. (2008); Damager, I. et al. (2008); and Buchini, S. et al. (2008)). The most probable mechanism for the GH34 sialidase (i.e. viral sialidases) reported in the literature is one involving an ion-pair intermediate (von Itzstein M. (2007)).
A number of compounds are known to inhibit NAs. Some well known NA inhibitors are alkene-containing sialic acid analogues (for example: Laninamivir CAS #203120-17-6; Oseltamivir (Tamiflu) CAS #204255-11-8; and Zanamivir (Relenza) CAS #139110-80-8; see also U.S. Pat. No. 5,360,817; and Ikeda et al. Bioorganic & Medicinal Chemistry (2006) 14:7893-7897). Fluorinated sugar derivatives with (reactive) fluoride leaving groups have been shown to be inhibitors of a range of “retaining” glycosidases and function via formation of particularly stable glycosyl-enzyme intermediates (for example, Zechel and Withers ((2000) Accounts of Chemical Research 33, 11-18) and Buchini et al. (2008)). These reagents are quite specific with respect to their target enzymes, have been shown to be highly bio-available, and even capable of crossing the blood-brain barrier. Such inhibitors are mechanism-based in their action, making the development of resistance by viruses difficult, whereby any mutations in the viral enzyme that reduce the inhibition must necessarily reduce the efficiency of the enzyme on the natural substrate, sialic acid and therefore less likely to be tolerated. The initial design of oseltamivir and zanamivir was based upon the mimicry of the flattened transition state conformation of the sugar through incorporation of an endocyclic alkene within a carbocycle (oseltamivir) or a pyranose ring (zanamivir) (M. von Itzstein et al., Nature 363, 418 (1993)). Specificity for the influenza enzyme over other NAs, along with additional affinity, was provided by incorporation of a guanidinium or ammonium substituent at the position corresponding to C-4 of the natural substrate to interact with a highly conserved anionic pocket at that location in the active site. These broad spectrum influenza drugs are active against NAs from group 1 and 2 influenza A strains as well as influenza B.
Despite being transition state analogue inhibitors, the emergence of drug-resistant strains has been reported, particularly against the more widely used and structurally divergent drug oseltamivir. Mutations can be both drug- and influenza subtype-specific. The most commonly seen mutation in viruses with the N1 subtype is H275Y which interferes with binding of the isopentyl side chain of oseltamivir, but still permits binding of zanamivir and the natural substrate. Mutations most commonly detected in clinical isolates with the N2 subtype include R292K (J. L. McKimm-Breschkin et al., J. Virol. 72, 2456 (1998); M. Tashiro et al., Antivir. Ther. 14, 751 (2009); M. Kiso et al., Lancet 364, 759 (2004)) and E119V (M. Tashiro et al., Antivir. Ther. 14, 751 (2009); M. Kiso et al., Lancet 364, 759 (2004)). Like the H275Y, the R292K precludes full rotation of the E276 necessary to create the hydrophobic pocket that accommodates the pentyl side-chain of oseltamivir (J. N. Varghese et al., Structure 6, 735 (1998)). In contrast, E119V confers oseltamivir specific resistance due to altered interactions with the 4-amino group. E119A, D, G mutations seen in vitro (T. J. Blick et al., Virology 214, 475 (1995); L. V. Gubareva et al., J. Virol. 71, 3385 (1997)) affect binding of oseltamivir and/or zanamivir, demonstrating the significance of the interactions of C-4 amino or guanidino group for high affinity binding. Some of the recent mutations seen in pandemic H1N1 viruses, including I223R, confer reduced sensitivity to both inhibitors (A. Eshaghi et al., Emerg. Infect. Dis. 17, 1472 (2011); H. T. Nguyen et al., Clin. Infect. Dis. 51, 983 (2010); E. van der Vries et al., N. Engl. J. Med. 363, 1381 (2010)). The emergence of mutant strains suggests that new neuraminidase inhibitors with increased propensity to maintain potency against such mutant viral strains would be of interest.